The nontuberculous Mycobacteria (NTM) are a growing public health concern as the number of opportunistic infections increases. Although not a reportable disease, there is growing epidemiologic evidence to suggest that NTM cause more infections today in the United States than Mycobacterium tuberculosis (Mtb) yet, unlike Mtb, there are few dedicated antimicrobial drug discovery programs specifically for NTM. Pulmonary disease caused by NTM is especially problematic in patients with underlying susceptibilities such as immunosuppressive medications, cystic fibrosis and other lung diseases, HIV and malignancies. Given the emergence of NTM as a public health issue, finding new antibacterial agents is of high importance. We have screened libraries of novel compounds for anti-NTM activity, focusing on M. abscessus (Mab), and identifying several promising small molecule screening hits with chemically-tractable scaffolds. Our Phase I SBIR grant focused on initial medicinal chemistry optimization of early drug leads that exhibited minimal cytotoxicity, high metabolic stability and low potential for resistance development. We achieved minimum inhibitory concentration (MIC) values ranging from 0.06 to 2 g/mL for rapidly growing Mycobacteria (RGM), including Mab, M. chelonae, M. fortiutum and M. peregrinum. Importantly, these compounds also have activity against Mtb (MIC 4 to 16 g/mL) and against permeabilized Gram-negative bacteria (GNB), potentially enabling us to attain broad spectrum antibacterial activity. We propose to continue development of a very promising benzothiazole cyclohexylcarboxamide series with Phase II SBIR support, focusing on advanced medicinal chemistry lead optimization with a goal of obtaining a strong drug candidate with demonstrated in vivo activity. In Aim 1, we will synthesize over a hundred analogs, which will be characterized in Aim 2 using a cascade of screening assays, beginning with MIC testing against RGM, followed by analysis of efflux, serum-binding, hemolysis, cytotoxicity, metabolic stability, frequency of resistance emergence, and microbiological spectrum including Mtb and GNB. Promising analogs will be screening for murine pharmacokinetic characteristics using an abbreviated pharmacokinetic protocol. The goal of Aims 1 and 2 will be to identify multiple lead compounds suitable for in vivo efficacy and toxicity testing during Aim 4. Aim 3 will establish mode of action, based on preliminary data indicating that the compounds target MmpL3, an essential protein thought to be involved in translocation of mycolic acids to or across the periplasmic space. Defining the mechanism will facilitate biochemical potency studies, thereby supporting the medicinal chemistry effort in Aim 1. In Aim 4, we will scale up synthesis of several lead compounds, begin to develop process chemistry strategies to improve synthetic efficiency, and evaluate chemical stability. We will utilized NIH preclinical services for the evaluation of maximum tolerated dose as well as efficacy in acute and chronic murine models of Mycobacterial infection. Successful completion of Aim 4 would bring this exciting chemical series to advanced preclinical development, setting the stage for identification of a candidate compound for clinical development. We believe this program holds great promise for development of a novel therapeutic agent with broad anti-mycobacterial activity.